Process for detection of biological activity

ABSTRACT

A sample of blood to be tested for the presence of biological activity is added to a suitable growth medium which includes C14 containing carbon source fermentable to produce gaseous C14O2. After a suitable incubation period, a portion of the gaseous atmosphere above the medium is analyzed for the presence of radioactivity. In the improvement herein described, a growth medium containing at least about 5 percent by weight of a material selected from the group consisting of sucrose, raffinose and glycylglycine is employed in order to reduce the incidence of high background readings due to sterile blood.

Unite States Patent 1 91 Waters et a1.

[451 Dec. 10, 1974 A. Zwarun, 7 Dell Ct., Baltimore, Md. 21207 22 FiledzDec. 3, 1973 21 App1.N0.:421,354

Related US. Application Data [63] Continuation-in-part of Ser. No.353,952, April 24,

[52] 11.8. C1 250/303, 250/380, 424/1, [51] Int. Cl. ..G0lt1/00, Cl2kH10 [58] Field of Search 424/1; 195/127 [56] References Cited UNITEDSTATES PATENTS 3,506,402 4/1970 Simon 250/380 Axen et al. 424/1 Waters250/380 Primary Examiner-Harold A. Dixon 5 7 ABSTRACT A sample of bloodto be tested for the presence of biological activity is added to asuitable growth medium which includes C containing carbon sourcefermentable to produce gaseous C 0 After a suitable incubation period, aportion of the gaseous atmosphere above the medium is analyzed for thepresence of radioactivity. In the improvement herein described, a growthmedium containing at least about 5 percent by weight of a materialselected from the group consisting of sucrose, raffinose andglycylglycine is employed in order to reduce the incidence of highbackground readings due to sterile blood.

8 Claims, No Drawings PROCESS FOR DETECTION OF BIOLOGICAL ACTIVITYRELATED APPLICATIONS This application is a continuation-in-part ofapplication Ser. No. 353,952 filed Apr. 24, 1973 and entitled Processfor Detection of Biological Activity.

DESCRIPTION OF THE INVENTION This invention is directed to an improvedmeans for the detection of biological activity in blood.

Biological activity in blood, such as bacteremia (bacteria in blood), inthe past, presented a difficult detection problem. Culturing was donemanually and, of course, was a tedious procedure that requiredconsiderable periods of time until the physical presence of bacteria,fungi, or other microorganisms could be detected.

More recently, a radiometric technique for the detection of biologicalactivity in the blood has undergone clinical testing and has beenadopted for commercial practice. ln that method samples of blood areinoculated into a suitable growth medium that includes a C containingcarbon source, the inoculated medium is incubated for a suitable period,and a portion of the gaseous atmosphere is analyzed for while in thegaseous state. Such process is described, inter alia, in U.S. Pat. No.3,676,679 issued July 11, 1972; and in the articles Early Detection ofBacterial Growth, with Carbon Labeled Glucose," Radiology, 92, No. 1,pp. l545 (Jan. 1969); Automated Radiometric Detection of BacterialGrowth in Blood Cultures," J. Labs. Clin. Med., 75, No. 3, pp. 529-34(March 1970); and Automated Radiometric Detection of Bacteria in 2,967Blood Cultures, Applied Microbiology, 22, No. 5, pp. 846-849 (Nov.1971). A commercialinstrument for the practice of a rapid, automatedprocess is available under the trademark Bactec (Johnston Laboratories,Inc.).

In practicing the process for the gaseous detection of the C O a generalbackground reading of about five units is frequently encountered. It hasalso been generally observed when the gas from a sample inoculated withblood is tested, the background reading is generally increased by tounits. In other words, even when the blood sample is from a patient whohas no microbial infection, the radioactivity will read, on the average,between about 15 and units. The gas from the blood sample of a patientwith bacteremia or the like, of course, provides an even higher reading.Generally, a reading of about units or more is used as an indication ofmicroorganisms in the blood sample. On occasion the blood backgroundreading can exceed 30 units making it very difficult to distinguish atrue positive caused by microbial infection from the background reading.

Reference to units, herein, is to units of the Bactec instrument. Onehundred units are equal to 0.025 microcuries of C activity.

While the described system has proven effective for the rapid detectionof biological activity in the blood and is in commercial use, it isdesirable to reduce the background readings due to blood andparticularly to reduce the incidence of high background readingsproduced by sterile blood.

It is an object of this invention to reduce the incidence of highbackground readings due to sterile blood.

It is another object of this invention to reduce the background readingin the gas measurement of the C 0 when blood is screened for thepresence of biological activity.

This invention is directed to the process of detecting biologicalactivity in the blood wherein:

a. a growth medium including a C containing carbon source which isfermentable to produce carbon dioxide is inoculated with a blood sample;

b. the inoculated sample is exposed to conditions conducive to theoccurrence of normal metabolic processes for a period of time sufficientto cause production of carbon dioxide by the fermenting of said source;and

c. at least a portion of the gaseous atmosphere from said fermentationis measured for radioactivity to determine the presence of C 0 Theimprovement, according to the present invention, comprises employing agrowth medium containing at least about 5 percent by weight of amaterial selected from the group consisting of sucrose, raffinose andglycylglycine, whereby the high background readings for the measurementof gaseous C 0 are reduced.

The precise reason for the increased background reading due tocomponents in the blood is not fully understood. Whatever the cause, ithas been discovered that the background reading is decreased if thegrowth medium contains at least about 5 percent of the additives of thisinvention. The mechanism by which such additives decrease the backgroundreading is not clear. While the additives of this invention increaseosmotic pressure, other agents which also increase osmotic pressure,such as dextran, lactose, sodium chloride, or potassium chloride, do notmaterially reduce the background reading caused by blood.

In tests employing this invention, the background reading due to theblood has generally been decreased somewhat; Importantly, however,anomalously high readings due to sterile blood may be eliminated so thatsuch samples produce only normal readings. Therefore the accuracy of thetechnique is increased.

The process to which this invention relates and the apparatus forcarrying out the process are described, inter alia, in US. Pat. No.3,676,679; Radiology, 92, No. 1, pp. 154-5 (Jan 1969); J. Labs. Clin.Med, 75, No. 3, pp. 529-34 (March 1970); Applied Microbiology, 22, No.5, pp. 846-849 (Nov. 1971); Johnston Laboratories, Inc. Technical DataApplication Notes JLI512,JL1604,JLI609A,JL1610A,JLI6l1,and JLI 612.These publications are incorporated herein by reference.

Generally, the process employs a nutrient medium that contains water, asuitable C containing carbon source, a nitrogen source, minerals andtrace elements. Typical C containing carbon sources may be glucose,fructose, galactose, mannose, rhamnose, or the like, phenylalanine,lysine, arginine or the like, glycerol, urea, or carboxylic acids suchas citric acid or the like. Glucose, which is readily available,constitutes a preferred C containing material. Generally the levels ofradioactivity will vary from about 0.1 to about 10 microcuries per 10ml. The assimilable nitrogen source may be either organic or inorganic,such as nitrates, nitrites, ammonia, urea, amino acids, or the like,while minerals such as the chlorides, sulfates or phosphates of calcium,sodium, potassium, magnesium or the like and trace elements such asmanganese, iron, zinc, cobalt or the like, may also be employed.Vitamins, cofactors or other enrichment agents such as anti-coagulantsmay also be added if desired. Finally, the medium may also include abuffer for pH adjustment and maintenance. The atmosphere above theculture medium can be air, oxygen, or the like if aerobic tests arebeing conducted, whereas nitrogen, CO or the like can be employed ifanaerobic tests are being conducted.

Commercial media (except for the additive of this invention) aredescribed in detail in the above publications and particularly in JLI 611, and JLI 612. A broad selection of possible components that may beincluded in the media is also set out in US. Pat. No. 3,676,679. Whilethat patent indicates that up to 20 percent or more carbohydrate may beemployed in the medium, preferred media are said to contain only up toabout 0.001 percent carbohydrate and commercial media do contain suchsmall amounts of carbohydrate added as such. In addition, if the mediacontains peptone, yeast extract, or the like, an additional 1 percent orso of carbohydrates may be present as a component of this additive.

Vials containing media for aerobic cultures, designated No. 6A, andvials containing media for anaerobic cultures, designated No. 7A, arecommercially available from Johnston Laboratories, Inc. A mediumsuitable for aerobic fermentation (6A) may contain tryptic soy broth,hemin, menadione, sodiumpolyanethol sulfonate, and C -labelledsubstrates, while a medium suitable for anaerobic culture (7A) maycontain tryptic soy broth, yeast extract, hemin, menadione, L-cysteine,sodium polyanethol sulfonate and C-labelled substrates. The 50 ml vialscontaining 30 ml of medium have a radioactivity of about 1.5microcuries. Commercial media have a pH of about 7.3.

Most frequently the sample of blood will be added to the medium inamounts ranging from about 0.5 to about 10 percent by volume and theinoculated medium can be cultured under agitation at temperaturesbetween about 20C and about 60C and most often between about 33 andabout 39C. The length of time for the culture will depend, inter alia,on the amount of inoculum employed and the particular microorganisminvolved. Tests have demonstrated that microorganisms generally willcreate a positive response in time periods ranging from 1 to 24 hours.

After a suitable period of time, e.g., 8 hours, the gaseous atmosphereover the culture medium is tested for radioactivity. Readings of about30 units or more generally have indicated the presence of microbialactivity in the blood. The threshold reading will, of course, depend inpart on the background reading due to the general surroundings. Suchnoise can readily be determined by testing the atmosphere from a mediumthat has not been inoculated. The reading used to indicate the presenceof microorganisms may, of course, vary somewhat depending on the levelof general background noise. When the threshold level has been reached,the sample is considered positive. Negative samples are furtherincubated and tested again to insure against the presence of slowgrowing microbes.

In accordance with the improvement of this invention, a materialselected from the group consisting of sucrose, raffinose, andglycylglycine is incorporated in the medium in amounts of at least about5 percent by weight and preferably at least about 10 percent by weight.Amounts of 30 percent by weight or more may be employed. Sucrose is aparticularly preferred additive of this invention. Because the additivesof this invention particularly reduce the high background readings dueto sterile blood, the threshold reading to indicate the presence ofmicrobes may be reduced to about 20 or about 25.

The following examples are included in order to illustrate the practiceof this invention. These examples are included for illustrative purposesonly and in no way are intended to limit the scope of the invention.

EXAMPLES l7 Stock culture bacteria were grown overnight on chocolateagar slants at 35C. The organisms tested were Diplococcus pneumoniae,I-Iaemophilus sp., Pseudomonas aeruginosa, Pseudomonas diminuta, andStreptococcus pyogenes. The growth on the slants was suspended with 1 mltryptic soy broth, diluted with tryptic soy broth to 1110,000 of theoriginal concentration, and 1 ml of each suspension inoculated into thetest media. For the Haemophilus culture, 1 ml of whole blood was alsoadded to the culture media. The control medium was a 50 ml JLI No. 6Ablood culture vial (Johnston Laboratories, Cockeysville, Maryland)containing 30 ml of enriched tryptic soy broth with 1.5 microcuries of C-labeled substrates and a magnetic stirring bar. The hypertonic mediaconsisted of various agents added to the control medium. These agentswere either 1% NaCl, 3% NaCl, 10% sucrose, 5% dextran (Sigma ChemicalCompany, St. Louis, Mo., approximate average molecular weight 15,00020,000), 10% dextran, or 15% dextran. All percentages are expressed on aweight to volume basis.

The evolution of C 0 by these cultures was monitored by using theautomated BACTEC Model 225 system (Johnston Laboratories). Thisradiometric technique has been demonstrated to be very sensitive indetecting C 0 production by all levels of bacterial inocula and is usedin clinical laboratories for routine blood culturing. The culture vialswere incubated at 35C while providing continuous agitation through themagnetic stirring bar. Samples were analyzed for C 0 production every 2hours using air 10% CO as the flushing and culture gas.

In Example 1, 1 ml of whole blood obtained from a healthy donor was usedinstead of bacteria. These blood cultures were tested as described aboveexcept on a 4-hour cycle.

Detection time of the cultures was taken when the Growth Index reached avalue of 20 or higher. The Growth Index (G1) is an arbitrary scale of 0to which is linearly proportional to the amount of C detected. Afull-scale reading of 100 corresponds to 0.025 microcurie of Cliberated. The maximum G1 indicated in the data is the highest GIproduced by a culture after its detection.

Table 1 provides some chemical characteristics of the agents and theresultant media, assuming 100 percent dissociation of NaCl.

TABLE 1 the sample was inoculated into a vial having 30 ml of JLl 6Amedium containing 10 percent sucrose. The moles particles} pH of maximumGrowth Index values for 103 clinical tests Ex Agent Aw/v Molurity litermedium are mponcd i T b 4 g 1 Control 7.4 2 NaCl 1 0.17 0.34 7.35 3 NaCl3 0.51 1.02 7.3 TABLE 4 4 sucrose 10 0.29 0.29 7.3 5 dextran 5 0.00250.0025 7.25 Max. Max. Max. 6 dextran 10 0.005 0.005 7.15 G1 G] G] 7dextran 15 0.0075 0.0075 7.05 10 -20 21-30 31 or more Control 85 15 3Control 107: sucrose 103 0 0 Table 2 reflects the effect of each of theagents on Th whole blood from a healthy patient. As this table dem- 15 fGI for 103 Control Samples onstrates, only the addition of 10% sucroseaffected the was Wh'le the f mfixlmum GI for the P maximum Growth Indexcent sucrose-containing med1um was 13.5. The median max1mum GI for the85 control samples in the O-2O category was while the median maximum G1for the TABLE 2 20 same 85 samples in 10 percent sucrose-containingmedium was 13.5.

Ex Medium Max. C11 EXAMPLE 9 C 7 The procedure of Examples 1-7 wasrepeated except 1 ontrol 2 comm 10% sucrose 8 that hemm and menadtonewere omitted from the me 3 C0mml+1% ct 15 dium and 3 ml of sterile bloodwas used as tnoculum. 4 Control+-3% NaCl 15 The results (mean of 3replicates) of the control, the. 5 Control+-5% dextran 20 Comr l t 1 6Control+10%dextran 20 O q percent sucrose, and 116 C01!- 7 Control+ 15%deman 22 trol containing 10 percent glycylglycine are shown in Table 5below.

TABLE 5 Medium 1 2 3 4 (days) Control 17 18 16 15 Table 3 represents theeffects of the varlous agents Control 10% sucrose 13 t0 9 8 on the tesbacteria Control 10% glycylglycine l7 l4 9 6 TABLE 3 D. neumoniae P.aeruginosa P. diminuta Haemophilgs S. gygggngsdetecdetecdetecdetecdetection tion tion tion tion k g k time max. timemax. time rriax. time max. time max. Ex (hrs) G1 (hrs) G1 (hrs) GI (hrs)G1 (hrs.) G1

1 Control 6 93 2 I00 14 49 6 100 4 32 2 Control+ 1% NaCl 8 76 2 100 1840 10 4 34 3 Control +3% NaCl NG 4 I00 PG NG 6 37 4 Control+10% sucrose10 55 4 [00 a 18 39 12 33 12 5 Control 5% dextran 8 4 100 16 45 6 66 637 6 Control+ 10% dextran 8 I00 4 100 18 40 6 61 6 28 7 I0 100 4 100 1844 6 67 8 28 Control 15% dextran NG, no growth; PG, poor growth; goodgrowth, no detection EXAMPLE 8 A 6 ml sample of blood was drawn from apatient and 6 3 ml of the sample was inoculated into a vial containing30 ml of .lLl 6A medium while the remaining 3 ml of EXAMPLES 10-16 aboutl.5 microcuries per 30 cc of medium. ln these examples. 50 cc vialscontaining 30 cc of the control medium were employed. The chemical andphysical characteristics of the media are shown in Table 6 be- We claim:l. [n the process of detecting biological activity in blood wherein:

a. a growth medium including a C containing carlow. bon source which isfermentablc to produce carbon TABLE 6 Ex Medium pH Appearance l0Control, tryptic soy broth with 0.025% sodium 7.2 clear solution Tpolyanethol sulfonate and the C-l4 sub stratesl.5 microcuiies per 30 cc,50 cc vial. l l Control+ 10% D Raffinose 7.2 clear solution 12 Control10% sucrose 7.2 clear solution 13 Control 10% D Galactose 6.0 very darksolution 14 Control l0% Glycl-L-asparagine 6.4 dark suspension 15Control+ l0% D Cellobiose 6.5 dark solution 16 Control+ 10% Lactose 6.6dark solution Three cc of sterile whole blood were inoculated m each ofthe media above. The vials were tested for CO periodically using amixture of 90 percent air and 10 percent CO as the flushing gas at C.The vials in these examples, as in the earlier examples, were maintainedat 35C. Replicates were run. The average maximal reading obtained isshown in Table 7 below.

TABLE 7 dioxide is inoculated with a blood sample;

b. the inoculated sample is exposed to conditions conducive to theoccurrence of normal metabolic processes for a period of time sufficientto cause production of carbon dioxide by the fermenting of said source;and

c. at least a portion of the gaseous atmosphere from Average Maximal GlObtained Average Max. Gl

As the readings (CI) reported in Table 7 d emonstrate, both sucrose andraffinose reduced the background noise.

EXAMPLE 17 TABLE 8 Sucrose Gl ll ll l0 pH 7.2 pH 6.8 pH 6.6

Since modification of this invention will be apparent to those skilledin the art, it is intended that this invention be limited only by thescope of the appended claims.

said fermentation is measured for radioactivity to determine thepresence of C 0 the improvement comprising incorporating into the mediumat least about 5 percent by weight of a compound selected from the groupconsisting of sucrose, raftinose and glycylglycine whereby thebackground noise for the measurement of radioactivity of gaseous C 0 isreduced.

2. The process of claim 1 wherein the compund is present in the mediumin amounts of at least about 10 percent by weight;

3. The process of claim 1 wherein the compound is sucrose;

4. The process of claim 2 wherein the compound sucrose;

5. The process of claim 1 wherein the compound raffmose;

6. The process of claim 2 wherein the compound raffinose;

7. The process of claim 1 wherein the compound glycylglycine;

8. The process of claim 2 wherein the compound glycylglycine.

1. IN THE PROCESS OF DETCTING BIOLOGICAL ACTIVITY IN BLOOD WHEREIN: A. AGROWTH MEDIUM INCLUDING A C14 CONTAINING CARBON SOURCE WHICH ISFERMENTABLE TO PRODUCE CARBON DIOXIDE IS INOCULATED WITH A BLOOD SAMPLE;B. THE INOCULATED SAMPLE IS EXPOSED TO CONDITIONS CONDUCIVE TO THEOCCURRENCE OF NORMAL METABOLIC PROCESSES FOR A PERIOD OF TIME SUFFICIENTTO CAUSE PRODUCTION OF CARBON DIOXIDE BY THE FERMENTAING OF SAID SOURCE;AND C. AT LEAST A PORTION OF THE GASEOUS ATMOSPHERE FROM SAIDFERMENTATION IS MEASURED FOR RADIOACTIVITY TO DETERMINED THE PRESENCE OFC14O2, THE IMPROVEMENT COMPRISING INCORPORATING INTO THE MEDIUM AT LEASTABOUT 5 PERCENT BY WEIGHT OF A COMPOUND SELECTED FROM THE GROUPCONSISTING OF SUCROSE, RAFFINOSE AND GLYCYLGLYCINE WHEREBY THE BACGROUNDNOISE FOR THE MEASUREMENT OF RADIOCATIVITY OF GASES C14O2 IS REDUCED. 2.The process of claim 1 wherein the compund is present in the medium inamounts of at least about 10 percent by weight;
 3. The process of claim1 wherein the compound is sucrose;
 4. The process of claim 2 wherein thecompound is sucrose;
 5. The process of claim 1 wherein the compound israffinose;
 6. The process of claim 2 wherein the compound is raffinose;7. The process of claim 1 wherein the compound is glycylglycine;
 8. Theprocess of claim 2 wherein the compound is glycylglycine.